Methods Of Treating Inflammation Associated Airway Diseases And Viral Infections

ABSTRACT

The present disclosure provides methods of treating a pathogen-induced lung inflammation in a subject are provided in which an anti-S100A9 antibody is administered to a subject. Methods of treating a respiratory virus infection by administering an anti-S100A9 antibody are also provided.

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 62/099,376, filed on Jan. 2, 2015, theentire disclosure of which is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No.5R01AI083387, awarded by NIH. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS Web as an ASCII formatted sequence listing with a file named“245039_ST25.txt”, created on, Dec. 24, 2015, and having a size of 4.17kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Influenza, commonly known as “the flu,” is an infectious disease causedby the influenza virus. The most common symptoms include: a high fever,runny nose, sore throat, muscle pains, headache, coughing, and feelingtired. These symptoms typically begin two days after exposure to thevirus and most last less than a week. However, the cough may last formore than two weeks. Complications of influenza may include viralpneumonia, secondary bacterial pneumonia, sinus infections, andworsening of previous health problems such as asthma or heart failure.The lung disease severity following influenza A virus (IAV) infection isdependent on the extent of inflammation in the respiratory tract. Severeinflammation in the lung manifests in development of pneumonia.Therefore, it is critical to identify cellular factors and dissect themolecular/cellular mechanism controlling inflammation in the respiratorytract during IAV infection. There remains a need for compositions andmethods to reduce inflammation during influenza A infection.

SUMMARY

Certain embodiments are directed to methods for treatingpathogen-induced lung inflammation in a subject comprising administeringan antibody that neutralizes S100A9 activity (anti-S100A9 antibody) tothe subject. Administration of an antibody that neutralizes S100A9reduces or ameliorates lung inflammation and infection associated lungdisease. In certain aspects the pathogen is a respiratory virus,bacteria, or fungus. Respiratory viruses include, but are not limited toRespiratory Syncytial Virus (RSV), Influenza virus (types A, B, C),Parainfluenza viruses (human prainfluenza viruses type I, II and III),Adenoviruses, Rhinoviruses, Human metapneumoviruses, and Coronaviruses(e.g. SARS). Respiratory bacteria and fungus include, but are notlimited to Streptococcus pyogenes (Group A), Haemophilus influenza,Bordetella pertussis, Moraxella catarrhalis, Streptococcus pneumoniae(pneumococcus), Staphylococcus aureus, Legionella pneumophila,Klebsiella pneumonia, Pseudomonas aeruginosa, Burkholderia cepacia,Mycoplasma pneumonia, Mycobacterium tuberculosis, Chlamydia Pneumoniae,Candida albicans, Coccidioides immitis, Histoplama capsulatum,Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillusfumigatus. In certain aspects the pathogen is an influenza virus. In afurther aspect the influenza virus is influenza A virus. In certainaspects the method can further comprise administering an antimicrobial(antiviral, antibiotic, or antifungal) drug in conjunction with theanti-S100A9 antibody.

In further aspects an antiviral drug is an amantadine, a neuraminidaseinhibitor (e.g., oseltamivir and zanamivir), ribavirin, and/orpalivizumab. In certain embodiments, the second therapeutic agent isselected from the group consisting of amantadine, oseltamivir, andzanamivir.

In certain aspects, the anti-S100A9 antibody is administeredintratracheally. Intratracheal administration can be in the form ofinstillation or inhalation of a composition comprising the anti-S100A9antibody. In certain aspects, the anti-S100A9 antibody is administeredvia a systemic administration. For example, a systemic administrationmay be via an intraperitoneal injection or an intravenous injection.

Certain embodiments are directed to methods and compositions comprisingneutralizing antibodies to S100A9 to reduce or ameliorate inflammationduring a respiratory infection. In certain aspects the infection is aninfluenza A infection. In certain embodiments the methods andcompositions can be used prophylactically in a subject at risk ofinfection or is susceptible to development of lung disease if infected,for example the elderly or immune suppressed subject.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be anembodiment of the invention that is applicable to other aspects of theinvention. It is contemplated that any embodiment discussed herein canbe implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIGS. 1A-1D. Production of S100A9 from IAV infected macrophages. U937cells (FIG. 1A), J774A.1 cells (FIG. 1B), primary bone marrow derivedmacrophages or BMDM (FIG. 1C) and primary mouse alveolar macrophages(FIG. 1D) were infected with IAV. U937 cells were infected at 1 MOI,whereas J774A.1, BMDM and primary alvelolar macrophages were infected at2 MOI. At indicated post-infection time-periods the medium supernatantwas collected to assess levels of S100A9 protein by ELISA. The valuesshown represent the mean±standard deviation from three independentexperiments performed in triplicate. *p<0.05 using a Student's t test.

FIGS. 2A-2F. DDX21 and TRIF are required for S100A9 production from IAVinfected macrophages. (FIG. 2A) Primary bone marrow derived macrophagesor BMDMs isolated from wild-type (WT), TLR2 knockout (KO), TLR4 KO andTRIF KO mice were infected with IAV (2 MOI). At 24 hours post-infectiontime-period the medium supernatant was collected to assess levels ofS100A9 protein by ELISA. (FIG. 2B) RT-PCR analysis of S100A9 expressionin IAV infected WT and TRIF KO BMDMs. (FIG. 2C) BMDMs isolated from WTand TLR3 KO mice were infected with IAV (2 MOI). At indicatedpost-infection time-periods the medium supernatant was collected toassess levels of S100A9 protein by ELISA. (FIG. 2D) Mouse alveolarmacrophage cell-line MH-S was transfected with either control siRNA orDDX21 specific siRNA. At 48 hours post-transfection, cells were infectedwith IAV (2 MOI). At indicated post-infection time-period RT-PCRanalysis was performed to examine expression of DDX21 in IAV infectedcontrol and DDX21 silenced cells. (FIG. 2E) MH-S cells transfected witheither control siRNA or DDX21 specific siRNA were infected with IAV (2MOI). At indicated post-infection time-period the medium supernatant wascollected to assess levels of S100A9 protein by ELISA. (FIG. 2F) BMDMsisolated from WT and TLR7 KO mice were infected with IAV (2 MOI). Atindicated post-infection time-periods the medium supernatant wascollected to assess levels of S100A9 protein by ELISA. The values shownin (FIG. 2A), (FIG. 2C), (FIG. 2E) and (FIG. 2F) represent themean±standard deviation from three independent experiments performed intriplicate. *p<0.05 using a Student's t test. Each RT-PCR data (FIG. 2Band FIG. 2D) is a representative of three independent experiments withsimilar results.

FIGS. 3A-3D. Extracellular S100A9 protein stimulates pro-inflammatoryresponse in macrophages. Human U937 macrophages were incubated withpurified recombinant human S100A9 protein (10m/ml) for 6 hours and 12hours. The medium supernatant was collected to assess levels of humanTNF-α (TNF) (FIG. 3A) and human IL-6 (FIG. 3B) by ELISA. Mouse J774A.1macrophages were incubated with purified recombinant mouse S100A9protein (5 μg/ml) for 6 hours and 12 hours. The medium supernatant wascollected to assess levels of mouse TNF (FIG. 3C) and mouse IL-6 (FIG.3D) by ELISA. The values represent the mean±standard deviation fromthree independent experiments performed in triplicate. *p<0.05 using aStudent's t test. Vehicle control cells (veh) were incubated with HBSSbuffer.

FIGS. 4A-4D. Extracellular S100A9 plays an essential role in inducingpro-inflammatory response during IAV infection of macrophages. (FIG. 4A)Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presenceof either control IgG (IgG) or anti-S100A9 blocking (neutralizing)antibody (S100A9 Ab). At indicated post-infection time-periods themedium supernatant was collected to assess levels of mouse IL-6 byELISA. (FIG. 4B) Primary bone marrow derived macrophages (BMDM) isolatedfrom wild-type (WT) and S100A9 knockout (KO) mice were infected with IAV(2 MOI). The medium supernatant was collected to assess levels of mouseIL-6 by ELISA. (FIG. 4C) WT and S100A9 KO BMDM were infected with IAV (2MOI). At the indicated post-infection time-period, medium supernatantwas collected to assess levels of mouse TNF-α (TNF) by ELISA. (FIG. 4D)S100A9 KO BMDMs were infected with IAV (2 MOI) in the presence ofpurified recombinant mouse S100A9 protein (5 μg/ml). Medium supernatantwas collected from infected cells to assess levels of mouse TNF and IL-6by ELISA. Vehicle control cells (veh) were incubated with HBSS buffer.The values represent the mean±standard deviation from three independentexperiments performed in triplicate. *p<0.05 using a Student's t test.

FIGS. 5A-5C. Extracellular S100A9 protein triggers apoptosis inmacrophages and S100A9 regulates apoptosis during IAV infection. (FIG.5A) Mouse J774A.1 macrophages were incubated with purified recombinantmouse S100A9 protein (5 μg/ml) for 48 hours and 72 hours. The apoptoticstate of these cells was examined by FACS analysis of annexin V and PIstained cells. Apoptosis rate (% apoptosis) was calculated based onnumber of annexin V positive/PI negative cells (denoting earlyapoptosis)+number of annexin V positive/PI positive cells (denoting lateapoptosis)/total number of cells. (FIG. 5B) Mouse alveolar macrophageMH-S cell-line was incubated with purified S100A9 protein (5 μg/ml) for48 hours and 72 hours. The apoptotic status was determined as describedin (FIG. 5A). (FIG. 5C) Mouse J774A.1 macrophages were infected with IAV(2 MOI) in the presence of either control IgG (IgG) or anti-S100A9blocking (neutralizing) antibody (S100A9 Ab). At 48 hourspost-infection, the apoptotic state of these cells was determined asdescribed in (FIG. 5A). The values (i.e. annexin V and PI stainingquantified by FACS) represents mean±standard deviation from threeindependent experiments, *p<0.05 by Student's t test. Veh; cellsincubated with HBSS buffer (vehicle control).

FIGS. 6A-6E. S100A9 activates TLR4/MyD88 pathway and activation ofTLR4/MyD88 pathway is essential for IAV-induced pro-inflammatoryresponse. Primary bone marrow derived macrophages (BMDM) isolated fromwild-type (WT) and TLR4 knockout (KO) mice were incubated with purifiedrecombinant mouse S100A9 protein (5 ug/mL). The medium supernatant wascollected to assess levels of mouse IL-6 (FIG. 6A) and mouse TNF-α (TNF)(FIG. 6B) by ELISA. (FIG. 6C) IL-6 production from S100A9 proteintreated WT and MyD88 KO BMDMs. (FIG. 6D) BMDM isolated from WT, TLR4 KOand MyD88 KO mice were infected with IAV (2 MOI). At 12 hours and 24hours post-infection time-period, medium supernatant was collected toassess levels of mouse IL-6 by ELISA. (FIG. 6E) TNF production from IAVinfected WT and TLR4 KO BMDMs. The values represent the mean±standarddeviation from three independent experiments performed in triplicate,*p<0.05 using a Student's t test. Veh; cells incubated with HBSS buffer(vehicle control).

FIGS. 7A-7D. Activated TLR4/MyD88 pathway promotes S100A9-mediatedapoptosis and is required for optimal apoptosis of IAV infected cells.(FIG. 7A) Primary bone marrow derived macrophages (BMDM) isolated fromwild-type (WT) and TLR4 knockout (KO) mice were incubated with purifiedrecombinant mouse S100A9 protein (5 ug/mL) for 72 hours. The apoptoticstate of these cells was examined by FACS analysis of annexin V and PIstained cells. Apoptosis rate (% apoptosis) was calculated based onnumber of annexin V positive/PI negative cells (denoting earlyapoptosis)+number of annexin V positive/PI positive cells (denoting lateapoptosis)/total number of cells. (FIG. 7B) WT and TLR4 KO BMDMs wereinfected with IAV (1 MOI). At 48 hours post-infection, the apoptoticstatus was determined as described in (FIG. 7A). (FIG. 7C) IAV infectedWT and TLR4 KO cells were subjected to TUNEL assay. TUNEL positive cellswere analyzed by image J software. Percent TUNEL positive cells denotesratio of number of TUNEL positive cells/total number of cells. (FIG. 7D)WT and MyD88 KO BMDMs were infected with IAV (1 MOI). At 48 hourspost-infection, the apoptotic status was determined. The valuesrepresents mean±standard deviation from three independent experiments,*p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer(vehicle control).

FIGS. 8A-8D. S100A9 expression and production in the IAV infectedrespiratory tract. (FIG. 8A) RNA isolated from mock infected and IAVinfected (2×104 pfu/mouse via intra-tracheal route) mice were subjectedto RT-PCR analysis to examine expression of mouse S100A9. The RT-PCRdata represents three mice/group (i.e. three mock mice, three miceinfected with IAV for 3 days, and three mice infected with IAV for 6days). The RT-PCR data is a representative of three independentexperiments with similar results. (FIG. 8B) Lung homogenate preparedfrom mock infected and IAV infected (2×104 pfu/mouse via intra-trachealroute) mice were subjected to ELISA analysis to determine levels ofmS100A9 protein in the lung. (FIG. 8C) Immuno-histochemical analysis ofmouse lung tissue sections derived from mock infected and IAV infectedmice were stained with mouse S100A9 antibody. Magnification, 200×. Onerepresentative example of a total of 3 mice analyzed per group in twoindependent experiments. (FIG. 8D) Broncho-alveolar lavage fluid (BALF)isolated from mock infected and IAV infected (2×104 pfu/mouse viaintra-tracheal route) mice were subjected to ELISA analysis to determinelevels of S100A9 protein in BALF. The values shown in (FIG. 8B) and(FIG. 8D) represent the mean±standard deviation from three independentexperiments performed in triplicate. *p<0.05 using a Student's t test.

FIGS. 9A-9E. S100A9 contributes to enhanced susceptibility andinflammation during IAV infection and S100A9 regulates pro-inflammatoryresponse in the respiratory tract of IAV infected mice. (FIG. 9A)Survival of IAV infected (1×105 pfu/mouse via intra-tracheal route) miceadministered with either control IgG (IgG) or anti-S100A9 blocking(neutralizing) antibody (S100A9 Ab) (24 hours prior to IAV inoculation,2 mg of antibody/mouse administered via i.p route). The data representsvalues from two independent experiments performed with 5 mice/group foreach experiment (total 10 mice/group from two experiments); *p=0.03.(FIG. 9B) Hematoxylin and eosin (H&E) staining of lung sections frommock infected or IAV infected mice (3×104 pfu/mouse via intra-trachealroute) administered with either control IgG (IgG) or S100A9 Ab (24 hoursprior to IAV inoculation, 2 mg of antibody/mouse was administered viai.p route). Magnification, ×10. (FIG. 9C) Mice were administered withpurified recombinant mouse S100A9 protein (15 μg/mouse) viaintra-tracheal route. At 8 hours post-administration, levels of mouseTNF-α in the lung was assessed by performing ELISA analysis with lunghomogenate. (FIG. 9D) Lung homogenate prepared from mock infected andIAV infected (2×104 pfu/mouse via intra-tracheal route) miceadministered with either control IgG (IgG) or anti-S100A9 blocking(neutralizing) antibody (S100A9 Ab) (24 hours prior to IAV inoculation,2 mg of antibody/mouse administered via i.p route) were subjected toELISA analysis to determine levels of mouse TNF-α in the lung. (FIG. 9E)For ex-vivo experiment, broncho-alveolar lavage fluid (BALF) wascollected (at 3 days post-infection) from IAV infected mice (2×104pfu/mouse via intra-tracheal route) mice administered with eithercontrol IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody(S100A9 Ab) (24 hours prior to IAV inoculation, 2 mg of antibody/mouseadministered via i.p route). The BALF cells were isolated and plated in48-well plate. After 2 hours and 4 hours, the medium supernatant wasanalyzed for mouse TNF-α (TNF) and mouse IL-6 by ELISA. Values shown in(FIG. 9C), (FIG. 9D) and (FIG. 9E) represent the mean±standard deviationfrom three independent experiments performed in triplicate. *p<0.05using a Student's t test. Veh; HBSS buffer diluted in PBS (vehiclecontrol).

FIGS. 10A-10C. Extracellular S100A9 promotes optimal apoptosis in thelung of IAV infected mice. (FIG. 10A) Lung sections were prepared (at 3days post-infection) from IAV infected (2×10⁴ pfu/mouse viaintra-tracheal route) mice administered with either control IgG (IgG) oranti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 hours priorto IAV inoculation, 2 mg of antibody/mouse administered via i.p route).For each experimental group lung sections were prepared from threecontrol IgG treated mice (+IAV) and three S100A9 Ab treated mice (+IAV).The lung sections were used for TUNEL staining. Image J software wasused to calculate TUNEL-positive areas (representing apoptosis) in thelung sections as detailed in the methods section. The data is presentedas percent apoptotic area. The percent apoptotic area was calculatedfrom nine areas/lung section as detailed in the methods section. Thevalues were compiled to calculate the percent apoptotic area in IAVinfected IgG treated mice vs. IAV infected S100A9 Ab treated mice,*p=0.0164 by Student's t test. (FIG. 10B) A representative TUNELstaining of lung sections from IAV infected mice administered witheither IgG or S100A9 Ab. The apoptotic nuclei (representing apoptosis)are indicated with red arrows. (FIG. 10C) A schematic model depictingthe role of extracellular S100A9 and DDX21/TRIF/S100A9/TLR4/MyD88signaling network in exaggerating lung disease during IAV infection. PM,plasma membrane; NM, nuclear membrane.

FIG. 11. RSV viral titer reduced in S100A9 Ab treated mice infected withRSV. Treatment with S100A9 blocking antibody (S100A9 Ab) demonstratedanti-viral properties to reduce RSV infection in the respiratory tractof mice compared to controls (treatment with IgG antibody).

FIG. 12. IL-6 production reduced in S100A9 Ab treated MH-S cellsinfected with RSV. Treatment of MH-S cells with S100A9 blocking antibody(S100A9 Ab) resulted in reduced IL-6 production following RSV infectioncompared to controls (treatment with IgG antibody).

DESCRIPTION

Influenza, commonly known as “the flu”, is an infectious disease causedby the influenza virus. Symptoms can be mild to severe. The most commonsymptoms include: a high fever, runny nose, sore throat, muscle pains,headache, coughing, and feeling tired. These symptoms typically begintwo days after exposure to the virus and most last less than a week. Thecough; however, may last for more than two weeks. In children there maybe nausea and vomiting but these are not common in adults. Nausea andvomiting occur more commonly in the unrelated infection gastroenteritis,which is sometimes inaccurately referred to as “stomach flu” or “24-hourflu”. Complications of influenza may include viral pneumonia, secondarybacterial pneumonia, sinus infections, and worsening of previous healthproblems such as asthma or heart failure. In the context of thisapplication influenza is used as an example of an inflammation inducingmicrobe. Embodiments are directed to pathogens that induce a similarresponse mediated by or including the S100A9 protein.

Additional inflammation associated airway diseases are also contemplatedto be within the scope of the present disclosure. For example, airwaydiseases commonly associated with an inflammatory component includepneumonia, bronchiolitis, chronic obstructive pulmonary disease (COPD),and asthma. The therapeutic compositions described herein are alsoapplicable to treatment of an inflammation associated airway disease.

Influenza viruses are RNA viruses that make up three of the five generaof the family Orthomyxoviridae: Influenza virus A, Influenza virus B,and Influenza virus C. These viruses are only distantly related to thehuman parainfluenza viruses, which are RNA viruses belonging to theparamyxovirus family that are a common cause of respiratory infectionsin children such as croup, but can also cause a disease similar toinfluenza in adults.

Influenza virus A has one species, influenza A virus. Wild aquatic birdsare the natural hosts for a large variety of influenza A. Occasionally,viruses are transmitted to other species and may then cause devastatingoutbreaks in domestic poultry or give rise to human influenza pandemics.The type A viruses are the most virulent human pathogens among the threeinfluenza types and cause the most severe disease. The influenza A viruscan be subdivided into different serotypes based on the antibodyresponse to these viruses. The serotypes that have been confirmed inhumans, ordered by the number of known human pandemic deaths, are: H1N1,H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H1ON7, and H7N9.

The Influenza virus B genus has one species, influenza B virus.Influenza B almost exclusively infects humans and is less common thaninfluenza A. This type of influenza mutates at a rate 2-3 times slowerthan type A and consequently is less genetically diverse, with only oneinfluenza B serotype.

The Influenza virus C genus has one species, influenza C virus, whichinfects humans, dogs and pigs, sometimes causing both severe illness andlocal epidemics. However, influenza C is less common than the othertypes and usually only causes mild disease in children

Pathogen-associated molecular patterns (PAMPs) trigger host immuneresponse by activating pattern recognition receptors like toll-likereceptors (TLRs). However, the mechanism whereby several pathogens,including viruses, activate TLRs via a non-PAMP mechanism is unclear.Endogenous “inflammatory mediators” called damage-associated molecularpatterns (DAMPs) have been implicated in regulating immune response andinflammation. However, the role of DAMPs in inflammation/immunity duringvirus infection has not been studied. We have identified a DAMPmolecule, S100A9 (also known as Calgranulin B or MRP-14), as anendogenous non-PAMP activator of TLR signaling during influenza A virus(IAV) infection. S100A9 was released from undamaged IAV-infected cellsand extracellular S100A9 acted as a critical host-derived molecularpattern to regulate inflammatory response outcome and disease duringinfection by exaggerating pro-inflammatory response, cell-death andvirus pathogenesis. Genetic studies showed that the DDX21-TRIF signalingpathway is required for S100A9 gene expression/production duringinfection. Furthermore, the inflammatory activity of extracellularS100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studieshave thus, underscored the role of a DAMP molecule (i.e. extracellularS100A9) in regulating virus-associated inflammation and uncovered apreviously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88signaling network in regulating inflammation during infection.

Protein S100A9 is also known as migration inhibitory factor-relatedprotein 14 (MRP-14) or calgranulin-B and is a protein that in humans isencoded by the S100A9 gene. S100A9 is a member of the S100 family ofproteins containing two EF hand calcium-binding motifs. S100 proteinsare localized in the cytoplasm and/or nucleus of a wide range of cells,and are involved in the regulation of a number of cellular processessuch as cell cycle progression and differentiation. S100 genes includeat least 13 members that are located as a cluster on chromosome 1q21.Human S100A9 (Unipro accession P06702) has an amino acid sequence ofMTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIEHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP (SEQ ID NO:15).

I. THERAPEUTIC COMPOSITIONS

Embodiments described herein are directed to a composition comprising anantibody that binds and neutralizes S100A9 ability to induceinflammation. Certain aspects are directed an antibody fragment orantibody conjugate, sufficient to treat or ameliorate pathogen-inducedinflammation or a complication thereof in a subject or a disease orcomplication associated with a pathogen infection in a subject whereinsaid antibody or antibody fragment or antibody conjugate bindsimmunospecifically to a S100A9. In certain aspects the pathogen is avirus. In certain aspects the virus is a respiratory virus, such asinfluenza. In a further aspect the pathogen is influenza A. Methods todetermine influenza A viral strains are well known to persons skilled inthe art. Therefore, influenza A viral strain variants (including, forexample, future mutations of influenza A strains and yet undeterminedinfluenza A viral strains) are within the scope of the instantdisclosure.

As used herein, the term “amount” refers to a concentration of antibodyor antibody fragment or antibody conjugate as determined by any meansknown to a skilled artisan, including weight of antibody, antibody titerof a unit dose, or a concentration of a unit dose of an antibody orantibody fragment or antibody conjugate. Preferably, an amount of anantibody or antibody fragment or antibody conjugate sufficient to treator prevent pathogen-induced inflammation or a complication thereof in asubject or diseases associated with a pathogen infection. In certainembodiments a subject is administered an amount of antibody or antibodyfragment that is sufficient to neutralize S100A9 activity as is itrelates to inflammation in an infected subject.

By “neutralize” is meant that the antibody or antibody fragment orantibody conjugate blocks the inflammation inducing capacity of S100A9.The precise amount of the antibody or antibody fragment or antibodyconjugate will vary depending on the specific activity of the antibodyor fragment and/or the purpose for which the composition is to be used.Accordingly, this term is not to be construed to limit the invention toa specific quantity, e.g., weight or concentration. Methods forassessing efficacy of any amount of an antibody or antibody fragment orantibody conjugate for treating or preventing virally inducedinflammation or complication thereof in a subject or a diseasesassociated with an infection will be apparent to the skilled artisanfrom the disclosure herein.

The term “antibody” means any protein or protein fragment having abinding domain with the required specificity and/or affinity for anepitope, including an immunoglobulin, antibody fragment, e.g., VH, VL,Fab, Fab′, F(ab)2, Fv, etc. Preferred “antibodies” within thisdefinition include intact polyclonal or monoclonal antibodies, animmunoglobulin (IgA, IgD, IgG, IgM, IgE) fraction, a chimeric antibody,a humanized antibody, an antibody fragment, or an immunoglobulin bindingdomain, whether natural or synthetic, and conjugates comprising same.Chimeric molecules including an immunoglobulin binding domain, orequivalent, fused to another polypeptide are also included within themeaning of the term “antibody” as used herein.

Anti-S100A9 antibodies, antibody fragments and antibody conjugates arereactive with an epitope of the S100A9 protein. For example, ananti-S100A9 antibody may be a blocking antibody or a neutralizingantibody. In certain aspects, antibodies are immunoglobulin fractions ormonoclonal antibodies or recombinant antibodies or humanized versionsthereof.

By “humanized antibody” is meant an antibody, antibody fragment orantibody conjugate comprising variable region framework residuessubstantially from, for example, a human antibody (termed an acceptorantibody) and complementarity determining regions substantially from,for example, a mouse-antibody, (referred to as the donorimmunoglobulin). Constant region(s), if present, is(are) substantiallyor entirely from a human immunoglobulin. The human variable domains areusually chosen from human antibodies whose framework sequences exhibit ahigh degree of sequence identity with a murine variable region domainfrom which the CDRs were derived. The heavy and light chain variableregion framework residues can be derived from the same or differenthuman antibody sequences. The human antibody sequences can be thesequences of naturally-occurring human antibodies or can be consensussequences of several human antibodies.

As used herein, the term “treat” or variations thereof such as“treatment” shall be taken to mean a treatment that ameliorates,reduces, or inhibits inflammation caused by a viral infection, orprevents or reduces the severity of one or more symptoms of a viralinfection. It is to be understood that such treatment therefore includesthe prophylaxis of a viral infection in so far as it prevents or reducessymptom development in an infected individual and/or preventsdevelopment of a complication thereof.

As used herein, the term “prophylactic treatment” refers to eitherpreventing or inhibiting the development of a clinical condition ordisorder or delaying the onset of a pre-clinically evident stage of aclinical condition or disorder. The term is to be understood as meaningthat the compositions according to the present invention can be appliedbefore symptoms of the infection are manifest. The compounds accordingto the present invention can, for example, be used in a prophylactictreatment.

The composition comprising an antibody, antibody fragment or antibodyconjugate that binds S100A9 as described herein is useful for treatingan infection or complication thereof in a human or other mammaliansubject or for treating a disease associated with infection of therespiratory system in a human or other mammalian subject. Preferably,the composition is for the treatment of humans.

Certain aspects provide a pharmaceutical composition comprising a doseof one or more antibodies, antibody fragments, or antibody conjugates asdescribed herein and a pharmaceutically acceptable carrier or excipient.Such compositions can be formulated for intratracheal or intranasal,delivery. Unit doses of antibody, antibody fragment, or antibodyconjugate can comprise from about 0.1m immunoglobulin per kilogram bodyweight to about 100 mg immunoglobulin per kilogram body weight, fromabout 0.1 m immunoglobulin per kilogram body weight to about 20 mgimmunoglobulin per kilogram body weight, from about 0.1 m immunoglobulinper kilogram body weight to about 10 mg immunoglobulin per kilogram bodyweight, or from about 0.1 m immunoglobulin per kilogram body weight toabout 1.0 mg immunoglobulin per kilogram body weight. Suitable carriersand excipients will vary according to the mode of administration andstorage requirements of a composition comprising an antibody, antibodyfragment, or antibody conjugate and are described herein.

As used herein, the term “suitable carrier or excipient” shall be takento mean a compound or mixture thereof that is suitable for use in acomposition for administration to a subject for the treatment of viralinfection or complication thereof in a subject or a disease orcomplication associated with a viral infection in a subject. Forexample, a suitable carrier or excipient for use in a pharmaceuticalcomposition for injection into a subject will generally not cause anadverse response in a subject.

A carrier or excipient useful in the pharmaceutical composition willgenerally not inhibit to any significant degree a relevant biologicalactivity of an antibody, antibody fragment, or antibody conjugate asdescribed according to any embodiment hereof, e.g., the carrier orexcipient will not significantly inhibit the ability of an antibody,fragment, or conjugate to bind to and neutralize S100A9. In certainaspects the carrier or excipient may include an antimicrobial compound.

Embodiments are directed to a method of treating or amelioratinginflammation due to an infection (e.g., an infection of the respiratorysystem) in a human or other mammalian subject. The method can compriseadministering to a subject having an infection or suspected of having aninfection or at risk of having an infection a composition as describedherein. The composition can be administered in an amount effective toprevent viral induced inflammation in a subject.

In an alternative embodiment, the present invention also provides amethod of preventing, ameliorating or treating a disease or complicationassociated with an infection or respiratory infection of a human orother mammalian subject. The method can comprise administering to thesubject a composition as described herein in an amount effective toreduce the severity of one or more disease symptoms or to prevent onsetof one or more diseases arising from the infection. In certain aspects,a method of treating a respiratory virus infection in a subject isprovided. The method comprises the step of administering an anti-S100A9antibody to the subject in need of thereof. In some embodiments, therespiratory virus is RSV. In other embodiments, the administration ofthe anti-S100A9 antibody reduces RSV infection in the respiratory tractof the subject via a decrease in viral titer.

A further embodiment is directed a method of neutralizing the activityof S100A9 in the lungs of a subject exposed to a pathogen. The methodcan comprise administering to a subject infected with a pathogen acomposition as described herein in an amount effective to reduce orneutralize S100A9 activity in the lung of the subject.

In certain embodiments, the therapeutic antibody, antibody fragment orimmunogenic moiety of an antibody conjugate is human or humanized, e.g.,an antibody wherein the human content of the antibody is maximized whilecausing little or no loss of binding affinity attributable to thevariable region of an antibody produced by a non-human antibody.

As discussed supra antibody fragments are contemplated. The term“antibody fragment” refers to a portion of a full-length antibody,generally the antigen binding or variable region. Examples of antibodyfragments include Fab, Fab′, F(ab′)2 and Fv fragments.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding.

The compositions and methods of the present invention may be used in thecontext of a number of therapeutic or prophylactic applications. Inorder to increase the effectiveness of a treatment with the compositionsof the present invention or to augment the protection of another therapy(a second therapeutic agent), e.g., antimicrobial therapy, it may bedesirable to combine these compositions and methods with other agentsand methods effective in the treatment, reduction of risk of infection,or prevention of diseases and pathologic conditions, for example,anti-bacterial, anti-viral, and/or anti-fungal treatments.

In certain aspects of the invention an anti-viral agent may be used incombination with a therapeutic composition described herein. Anti-viralagents include, but are not limited to abacavir; acemannan; acyclovir;acyclovir sodium; adefovir; alovudine; alvircept sudotox; amantadinehydrochloride; amprenavir; aranotin; arildone; atevirdine mesylate;avridine; cidofovir; cipamfylline; cytarabine hydrochloride; delavirdinemesylate; desciclovir; didanosine; disoxaril; edoxudine; efavirenz;enviradene; enviroxime; famciclovir; famotine hydrochloride;fiacitabine; fialuridine; fosarilate; trisodium phosphonoformate;fosfonet sodium; ganciclovir; ganciclovir sodium; idoxuridine;indinavir; kethoxal; lamivudine; lobucavir; memotine hydrochloride;methisazone; nelfinavir; nevirapine; palivizumab; penciclovir;pirodavir; ribavirin; rimantadine hydrochloride; ritonavir; saquinavirmesylate; somantadine hydrochloride; sorivudine; statolon; stavudine;tilorone hydrochloride; trifluridine; valacyclovir hydrochloride;vidarabine; vidarabine phosphate; vidarabine sodium phosphate; viroxime;zalcitabine; zidovudine; zinviroxime, interferon, cyclovir,alpha-interferon, and/or beta globulin. In certain aspects, otherantibodies against viral proteins or cellular factors may be used incombination with a therapeutic composition described herein.

Examples of anti-bacterials include, but are not limited to, β-lactamantibiotics, penicillins (such as natural penicillins, aminopenicillins,penicillinase-resistant penicillins, carboxy penicillins, ureidopenicillins), cephalosporins (first generation, second generation, andthird generation cephalosporins), and other β-lactams (such as imipenem,monobactams,), β-lactamase inhibitors, vancomycin, aminoglycosides andspectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin,clindamycin, rifampin, metronidazole, polymyxins, sulfonamides andtrimethoprim, and quinolines. Anti-bacterials also include, but are notlimited to: Acedapsone, Acetosulfone Sodium, Alamecin, Alexidine,Amdinocillin, Amdinocillin Pivoxil, Amicycline, Amifloxacin, AmifloxacinMesylate, Amikacin, Amikacin Sulfate, Aminosalicylic acid,Aminosalicylate sodium, Amoxicillin, Amphomycin, Ampicillin, AmpicillinSodium, Apalcillin Sodium, Apramycin, Aspartocin, Astromicin Sulfate,Avilamycin, Avoparcin, Azithromycin, Azlocillin, Azlocillin Sodium,Bacampicillin Hydrochloride, Bacitracin, Bacitracin MethyleneDisalicylate, Bacitracin Zinc, Bambermycins, Benzoylpas Calcium,Berythromycin, Betamicin Sulfate, Biapenem, Biniramycin, BiphenamineHydrochloride, Bispyrithione Magsulfex, Butikacin, Butirosin Sulfate,Capreomycin Sulfate, Carbadox, Carbenicillin Disodium, CarbenicillinIndanyl Sodium, Carbenicillin Phenyl Sodium, Carbenicillin Potassium,Carumonam Sodium, Cefaclor, Cefadroxil, Cefamandole, Cefamandole Nafate,Cefamandole Sodium, Cefaparole, Cefatrizine, Cefazaflur Sodium,Cefazolin, Cefazolin Sodium, Cefbuperazone, Cefdinir, Cefepime, CefepimeHydrochloride, Cefetecol, Cefixime, Cefinenoxime Hydrochloride,Cefinetazole, Cefinetazole Sodium, Cefonicid Monosodium, CefonicidSodium, Cefoperazone Sodium, Ceforanide, Cefotaxime Sodium, Cefotetan,Cefotetan Disodium, Cefotiam Hydrochloride, Cefoxitin, Cefoxitin Sodium,Cefpimizole, Cefpimizole Sodium, Cefpiramide, Cefpiramide Sodium,Cefpirome Sulfate, Cefpodoxime Proxetil, Cefprozil, Cefroxadine,Cefsulodin Sodium, Ceftazidime, Ceftibuten, Ceftizoxime Sodium,Ceftriaxone Sodium, Cefuroxime, Cefuroxime Axetil, Cefuroxime Pivoxetil,Cefuroxime Sodium, Cephacetrile Sodium, Cephalexin, CephalexiiHydrochloride, Cephaloglycini, Cephaloridine, Cephalothin Sodium,Cephapirin Sodium, Cephradine, Cetocycline Hydrochloride, Cetophenicol,Chloramphenicol, Cliloramphenicol Palmitate, ChloramphenicolPantotheniate Complex, Chloramphenicol Sodium Succinate, ChlorhexidinePhosphanilate, Chloroxylenol, Chlortetracycline Bisulfate,Chlortetracycline Hydrochloride, Cinoxacin, Ciprofloxacin, CiprofloxacinHydrochloride, Cirolemycin, Clarithromycin, Clinafloxacin Hydrochloride,Clildamycin, Clindamycin Hydrochloride, Clindamycin PalmitateHydrochloride, Clindamycin Phosphate, Clofazimine, CloxacillinBenzathine, Cloxacillin Sodium, Cloxyquin, Colistimethate Sodium,Colistin Sulfate, Coumermycin, Coumermycin Sodium, Cyclacillin,Cycloserine, Dalfopristin, Dapsone, Daptomycin, Demeclocycine,Demeclocycine Hydrochloride, Demecycline, Denofungin, Diaveridine,Dicloxacillin, Dicloxacillin Sodium, Dihydrostreptomycin Sulfate,Dipyrithione, Dirithromycin, Doxycycline, Doxycycline Calcium,Doxycycline Fosfatex, Doxycycline Hyclate, Droxacin Sodium, Enoxacin,Epicillin, Epitetracycline Hydrochloride, Erythromycin, ErythromycinAcistrate, Erythromycin Estolate, Erythromycin Ethylsuccinate,Erythromycin Gluceptate, Erythromycin Lactobionate, ErythromycinPropionate, Erythromycin Stearate, Ethambutol Hydrochloride,Ethionamide, Fleroxacin, Floxacillin, Fludalanine, Flumequine,Fosfomycin, Fosfomycin Tromethamine, Fumoxicillin, Furazolium Chloride,Furazolium Tartrate, Fusidate Sodium, Fusidic Acid, Gentamicin Sulfate,Gloximonam, Gramicidin, Haloprogin, Hetacillin, Hetacillin Potassium,Hexedine, Ibafloxacin, Imipenem, Isoconazole, Isepamicin, Isoniazid,Josamycin, Kanamycin Sulfate, Kitasamycin, Levofuraltadone,Levopropylcillin Potassium, Lexithromycin, Lincomycin, LincomycinHydrochloride, Lomefloxacin, Lomefloxacin Hydrochloride, LomefloxacinMesylate, Loracarbef, Mafenide, Meclocycline, MeclocyclineSulfosalicylate, Megalomicin Potassium Phosphate, Mequidox, Meropenem,Methacycline, Methacycline Hydrochloride, Methenamine, MethenamineHippurate, Methenamine Mandelate, Methicillin Sodium, Metioprim,Metronidazole Hydrochloride, Metronidazole Phosphate, Mezlocillin,Mezlocillin Sodium, Minocycline, Minocycline Hydrochloride, MirincamycinHydrochloride, Monensin, Monensin Sodium, Nafcillin Sodium, NalidixateSodium, Nalidixic Acid, Natamycin, Nebramycin, Neomycin Palmitate,Neomycin Sulfate, Neomycin Undecylenate, Netilmicin Sulfate,Neutramycin, Nifuradene, Nifuraldezone, Nifuratel, Nifuratrone,Nifurdazil, Nifurimide, Nifuirpirinol, Nifurquinazol, Nifurthiazole,Nitrocycline, Nitrofurantoin, Nitromide, Norfloxacin, Novobiocin Sodium,Ofloxacin, Ormetoprim, Oxacillin Sodium, Oximonam, Oximonam Sodium,Oxolinic Acid, Oxytetracycline, Oxytetracycline Calcium, OxytetracyclineHydrochloride, Paldimycin, Parachlorophenol, Paulomycin, Pefloxacin,Pefloxacin Mesylate, Penamecillin, Penicillin G Benzathine, Penicillin GPotassium, Penicillin G Procaine, Penicillin G Sodium, Penicillin V,Penicillin V Benzathine, Penicillin V Hydrabamine, Penicillin VPotassium, Pentizidone Sodium, Phenyl Aminosalicylate, PiperacillinSodium, Pirbenicillin Sodium, Piridicillin Sodium, PirlimycinHydrochloride, Pivampicillin Hydrochloride, Pivampicillin Pamoate,Pivampicillin Probenate, Polymyxin B Sulfate, Porfiromycin, Propikacin,Pyrazinamide, Pyrithione Zinc, Quindecamine Acetate, Quinupristin,Racephenicol, Ramoplanin, Ranimycin, Relomycin, Repromicin, Rifabutin,Rifametane, Rifamexil, Rifamide, Rifampin, Rifapentine, Rifaximin,Rolitetracycline, Rolitetracycline Nitrate, Rosaramicin, RosaramicinButyrate, Rosaramicin Propionate, Rosaramicin Sodium Phosphate,Rosaramicin Stearate, Rosoxacin, Roxarsone, Roxithromycin, Sancycline,Sanfetrinem Sodium, Sarmoxicillin, Sarpicillin, Scopafungin, Sisomicin,Sisomicin Sulfate, Sparfloxacin, Spectinomycin Hydrochloride,Spiramycin, Stallimycin Hydrochloride, Steffimycin, StreptomycinSulfate, Streptonicozid, Sulfabenz, Sulfabenzamide, Sulfacetamide,Sulfacetamide Sodium, Sulfacytine, Sulfadiazine, Sulfadiazine Sodium,Sulfadoxine, Sulfalene, Sulfamerazine, Sulfameter, Sulfamethazine,Sulfamethizole, Sulfamethoxazole, Sulfamonomethoxine, Sulfamoxole,Sulfanilate Zinc, Sulfanitran, Sulfas alazine, Sulfasomizole,Sulfathiazole, Sulfazamet, Sulfisoxazole, Sulfisoxazole Acetyl,Sulfisoxazole Diolamine, Sulfomyxin, Sulopenem, Sultamicillin, SuncillinSodium, Talampicillin Hydrochloride, Teicoplanin, TemafloxacinHydrochloride, Temocillin, Tetracycline, Tetracycline Hydrochloride,Tetracycline Phosphate Complex, Tetroxoprim, Thiamphenicol,Thiphencillin Potassium, Ticarcillin Cresyl Sodium, TicarcillinDisodium, Ticarcillin Monosodium, Ticlatone, Tiodonium Chloride,Tobramycin, Tobramycin Sulfate, Tosufloxacin, Trimethoprim, TrimethoprimSulfate, Trisulfapyrimidines, Troleandomycin, Trospectomycin Sulfate,Tyrothricin, Vancomycin, Vancomycin Hydrochloride, Virginiamycin, and/orZorbamycin.

Anti-fungal agents include, but are not limited to, azoles, imidazoles,polyenes, posaconazole, fluconazole, itraconazole, amphotericin B,5-fluorocytosine, miconazole, ketoconazole, Myambutol (EthambutolHydrochloride), Dapsone (4,4′-diaminodiphenylsulfone), Paser Granules(aminosalicylic acid granules), rifapentine, Pyrazinamide, Isoniazid,Rifadin IV, Rifampin, Pyrazinamide, Streptomycin Sulfate and Trecator-SC(Ethionamide) and/or voriconazole (VfendTM).

II. PHARMACEUTICAL COMPOSITIONS

Certain aspects include pharmaceutical compositions comprising atherapeutic antibody or antibody fragment. In a further aspect thecomposition can include other active molecules and one or morepharmaceutically acceptable carriers and/or diluents. The activeingredients of a pharmaceutical composition comprising an antibody canexhibit antiviral activity. For example, from about 0.1 μg to about 100mg per kilogram of body weight per day may be administered. Dosageregime may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation. The active compound may be administered in aconvenient manner such as by delivery to the respiratory system (e.g.,intratracheal or intranasal administration). Depending on the route ofadministration, the active ingredients which comprise an antibody orantibody fragment may be required to be coated in a material to protectsaid ingredients from the action of enzymes, acids and other naturalconditions which may inactivate said ingredients.

Pulmonary/respiratory drug delivery can be implemented by differentapproaches, including liquid nebulizers, aerosol-based metered doseinhalers (MDI's), sprayers, dry powder dispersion devices and the like.Such methods and compositions are well known to those of skill in theart, as indicated by U.S. Pat. Nos. 6,797,258, 6,794,357, 6,737,045, and6,488,953, all of which are incorporated by reference. According to theinvention, at least one pharmaceutical composition can be delivered byany of a variety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. Other devicessuitable for directing pulmonary or nasal administration are also knownin the art. Typically, for pulmonary administration, at least onepharmaceutical composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses. Some specificexamples of commercially available inhalation devices suitable for thepractice of this invention are Turbohaler™ (Astra), Rotahaler® (Glaxo),Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, AERx™ (Aradigm), the Ultravent® nebulizer (Mallinckrodt),the Acorn II® nebulizer (Marquest Medical Products), the Ventolin®metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), orthe like.

All such inhalation devices can be used for the administration of apharmaceutical composition in an aerosol. Such aerosols may compriseeither solutions (both aqueous and non aqueous) or solid particles.Metered dose inhalers typically use a propellant gas and requireactuation during inspiration. See, e.g., WO 98/35888; WO 94/16970. Drypowder inhalers use breath-actuation of a mixed powder. See U.S. Pat.Nos. 5,458,135; 4,668,218; PCT publications WO 97/25086; WO 94/08552; WO94/06498; and European application EP 0237507, each of which isincorporated herein by reference in their entirety. Nebulizers produceaerosols from solutions, while metered dose inhalers, dry powderinhalers, and the like generate small particle aerosols. Suitableformulations for administration include, but are not limited to nasalspray or nasal drops, and may include aqueous or oily solutions of atherapeutic composition as described herein.

A spray comprising a pharmaceutical composition as described herein canbe produced by forcing a suspension or solution of a composition througha nozzle under pressure. The nozzle size and configuration, the appliedpressure, and the liquid feed rate can be chosen to achieve the desiredoutput and particle size. An electrospray can be produced, for example,by an electric field in connection with a capillary or nozzle feed.

A pharmaceutical composition as described herein can be administered bya nebulizer such as a jet nebulizer or an ultrasonic nebulizer.Typically, in a jet nebulizer, a compressed air source is used to createa high-velocity air jet through an orifice. As the gas expands beyondthe nozzle, a low-pressure region is created, which draws a compositionthrough a capillary tube connected to a liquid reservoir. The liquidstream from the capillary tube is sheared into unstable filaments anddroplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the compositioncreating an aerosol.

It is advantageous to formulate compositions in dosage unit form forease of administration and uniformity of dosage. “Dosage unit form” asused herein refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated; each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms of apharmaceutical composition of the invention are dictated by and directlydependent on (a) the unique characteristics of the active material andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active materialfor the treatment of disease in living subjects having a diseasedcondition in which bodily health is impaired as herein disclosed indetail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in an amount ranging from about 0.1 μg to about 100 mg.In the case of compositions containing supplementary active ingredients,the dosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

III. EXAMPLES

The following examples as well as the figures are included todemonstrate preferred embodiments of the invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples or figures represent techniques discovered by theinventors to function well in the practice of the invention, and thuscan be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1

S100A9 secretion from IAV-infected macrophages. Macrophages areessential immune cells that modulate host defense, inflammation, anddisease pathogenesis during IAV infection. Macrophages are also themajor cytokine- and chemokine-producing cells during IAV infection andthus contribute to lung tissue damage. To investigate whether IAVinfection stimulates S100A9 secretion, macrophages were infected withIAV for 4-16 hours. After infection, medium supernatant was collected toassess S100A9 protein levels by ELISA. Following IAV infection bothhuman (U937 cells) (FIG. 1A) and mouse [J774A.1 macrophage cell-line,primary alveolar macrophages, and primary bone marrow-derivedmacrophages (BMDMs)] macrophages (FIG. 1B-1D) secreted high levels ofS100A9. The physiological significance is evident from the ability ofprimary macrophages (i.e. alveolar macrophages and BMDMs) (FIG. 1C and1D) to secrete S100A9 upon IAV infection. Interestingly, S100A9secretion was detected as early as 4-8 hours postinfection. Release ofS100A9 is not due to cell cytotoxicity or damage, since an LDH releasecytotoxicity assay showed minimal cytotoxicity in macrophages at 8 and16 hours postinfection. Similarly, no cell death (apoptosis or necrosis)was observed during the 8-16 hours postinfection period (not shown).These results demonstrated that following IAV infection, S100A9 isreleased to the extracellular environment from undamaged macrophages.

The DDX21/TRIF pathway is required for S100A9 gene expression andsecretion during IAV infection. There have been no studies to date onthe signaling mechanism that regulates gene expression of S100 family ofproteins. The signaling mechanism involved in S100A9 expression wasexamined. BMDMs derived from wild-type (WT), TLR2 knockout (KO), TLR4KO, and TRIF KO mice were infected with IAV. At 24 hours postinfection,evaluated S100A9 levels in the medium were evaluated. TLR2 and TLR4 werenot involved, since comparable S100A9 secretion was noted in WT and TLRKO BMDMs (FIG. 2A). A similar result was obtained with TRAM KO and TIRAPKO cells (not shown). In contrast, significant reduction in S100A9secretion was observed in IAV-infected TRIF KO BMDMs (FIG. 2A). RT-PCRanalysis showed that loss of S100A9 secretion was caused by the absenceof S100A9 mRNA in infected TRIF KO cells (FIG. 2B). Apart from TLR4,which uses TRIF for MyD88-independent signaling, TLR3 also recruits TRIFfor TLR3-mediated signal transduction. However, TLR3 is not involved inthis process, as shown by the fact that S100A9 secretion was not reducedin TLR3 KO BMDMs (FIG. 2C). These results demonstrated that S100A9 geneinduction occurs via the TLR-independent TRIF-dependent pathway.

Recently, DEAD box proteins (also known as DDX protein) having RNAhelicase activity has been implicated in innate immunity (Zhang et al.(2011) Immunity 34: 866-878). DDX proteins (e.g. DDX21) can function ascytosolic PRR in mouse dendritic cells (mDCs) to induce type-Iinterferon during infection (Zhang et al. (2011) Immunity 34: 866-878).Interestingly, DDX signaling was TRIF-dependent and DDX21 interactedwith TRIF during signaling (Zhang et al. (2011) Immunity 34: 866-878).Therefore, the inventors examined whether DDX21 has a role in S100A9expression during IAV infection of macrophages. Since KO animals lackingDDX proteins do not exist, siRNA technology was used to silence DDX21expression in macrophages.

Mouse alveolar macrophages (MH-S cell line) were transfected withDDX21-specific siRNA or control scrambled siRNA, after which these cellswere infected with IAV. DDX21 expression was monitored by RT-PCR.Induction of DDX21 expression was observed following IAV infection (FIG.2D). The silencing efficiency was evident from the loss of DDX21expression in IAV-infected cells transfected with DDX21-specific siRNA(FIG. 2D). The silenced cells were used to deduce the role of DDX21 inS100A9 gene expression following IAV infection. DDX21 is critical forS100A9 gene expression, since drastic loss of S100A9 mRNA was observedin IAV-infected DDX21 silenced cells. Accordingly, reduced S100A9 mRNAexpression in DDX21 silenced cells led to diminished S100A9 secretionfollowing IAV infection of these cells (FIG. 2E). The DDX/TRIF dependentS100A9 expression was independent of virus replication, since IAVhemagglutinin (HA) mRNA levels were similar in DDX21 silenced and TRIFKO cells. Moreover, S100A9 expression (not shown) and production was notsignificantly altered in IAV infected MyD88 KO and MAVS KO cells, whichimplicated MyD88 and MAVS independent expression/production of S100A9during IAV infection. In addition, the inventors failed to observesignificant difference in S100A9 expression/production from IAV infectedWT vs. TLR7 KO cells (FIG. 2F). It is interesting to note that DDX21expression was undetected at 48 hours postinfection (FIG. 2D), whichco-relates with loss of S100A9 production during that time frame (notshown). This suggests that to maintain homeostasis and to avoidhyper-inflammation cells may negatively regulate DDX21 expression toreduce S100A9 production. These results demonstrated that the DDX21/TRIFpathway is required for S100A9 gene induction and the resulting S100A9secretion following IAV infection.

Extracellular S100A9 promotes pro-inflammatory response during IAVinfection. In the preceding studies, the high levels of S100A9 secretionduring infection suggested that secreted extracellular S100A9 may havesome role during IAV infection. Therefore, the inventors focused on therole and function of extracellular S100A9 during IAV infection. Earlierstudies have shown that the S100A9/S100A8 complex is required foroptimal LPS-induced TLR4-dependent TNF-α (TNF) production in bone marrowcells (comprised of undifferentiated monocytes and DCs) (Vogl et al.(2007) Nat Med 13: 1042-1049). However, few studies have shown thepro-inflammatory activity of S100A9 in the absence of S100A8 and LPS.Moreover, it is not known whether S100A9 can launch a pro-inflammatoryresponse in macrophages. Since the studies are focused on the innateresponses of IAV-infected macrophages, the inventors investigatedwhether extracellular addition of purified S100A9 protein (to mimicsecreted S100A9) promotes the release of pro-inflammatory cytokines IL-6and TNF-α (TNF). These pro-inflammatory cytokines are produced earlyduring IAV infection, a period that corresponds with S100A9 secretionkinetics.

Mouse (J774A.1) and human (U937 cells) macrophages were incubated withpurified mouse or human S100A9 proteins, respectively for 6-12 hours(FIG. 3). After treatment, medium supernatant was collected to analyzeTNF and IL-6 levels by ELISA. S100A9 alone stimulates a pro-inflammatoryresponse in macrophages, as is evident from high levels of TNF (FIG. 3Aand 3C) and IL-6 (FIG. 3B and 3D) production by macrophages treated withpurified S100A9 protein. Both human (FIG. 3A and 3B) and mouse (FIG. 3Cand 3D) macrophages produced pro-inflammatory cytokines upon incubationwith human and mouse S100A9 protein. Interestingly, the response wasrapid, since substantial TNF and IL-6 production occurred within 6 hoursof treatment with S100A9 protein. RT-PCR analysis showed that productionof TNF and IL-6 by S100A9 was due to activation of their correspondinggenes. Since the pro-inflammatory activity of purified S100A9 proteincould be inhibited by anti-S100A9 blocking (neutralizing) antibody (notshown), the observed response was due to S100A9 protein. Moreover, theeffect observed with purified S100A9 protein was not due to LPScontamination. These studies demonstrated that S100A9 functions as anextracellular host factor to launch a pro-inflammatory response inmacrophages.

The inventors next examined the role of secreted S100A9 in eliciting apro-inflammatory response during IAV infection. A blocking antibodyagainst S100A9 was used to neutralize the activity of extracellular(secreted) S100A9. Previously, it was shown that this blocking antibodyspecifically inhibited the activity of the secreted extracellular formof S100A9 both in vitro and in vivo (Ryckman et al. (2003) J Immunol170: 3233-3242; Anceriz et al. (2007) Biochem Biophys Res Commun 354:84-89; Cesaro et al. (2012) PLoS One 7: e45478; Simard et al. (2010) JLeukoc Biot 87: 905-914; Simard et al. (2011) J Immunol 186: 3622-3631;Raquil et al. (2008) J Immunol 180: 3366-3374; Vandal et al. (2003) JImmunol 171: 2602-2609). J774A.1 cells were infected with IAV in thepresence of either control antibody (control IgG) or S100A9-specificblocking antibody. At various postinfection time points, IL-6 and TNFlevels were examined by ELISA. Extracellular S100A9 plays a key role ininducing the pro-inflammatory response during IAV infection, sincesignificant reduction in IL-6 (FIG. 4A) and TNF levels were observed ininfected cells treated with S100A9 blocking antibody. RT-PCR showed thatloss of IL-6 and TNF production was due to diminished gene expression(not shown). Similar results were obtained following treatment ofIAV-infected primary macrophages (BMDM) with S100A9 blocking antibody.Diminished IL-6 and TNF (not shown) production and expression wasobserved in infected BMDM treated with S100A9 blocking antibody. Theloss of pro-inflammatory response was not due to reduced IAVreplication, since IAV HA expression was similar in control antibody andS100A9 blocking antibody treated J774A.1 cells and BMDMs. Thus,extracellular S100A9 modulates pro-inflammatory response independent ofIAV replication.

The finding that S100A9 contributes to the pro-inflammatory responseduring IAV infection was validated by using BMDMs derived from S100A9 KOmice. WT and S100A9 KO BMDMs were infected with IAV, after which TNF andIL-6 levels in the medium supernatant were measured by ELISA. Ascompared to WT cells, there were significant reductions in IL-6 (FIG.4B) and TNF (FIG. 4C) production from infected S100A9 KO cells. Onceagain, this was a consequence of the loss of pro-inflammatory geneexpression in IAV-infected S100A9 KO BMDMs. The critical function ofsecreted (extracellular-form) S100A9 during this response was apparentfrom the observation that addition of purified mouse S100A9 protein toS100A9 KO BMDMs restored the pro-inflammatory response in IAV-infectedS100A9 KO BMDMs (FIG. 4D). This result also suggested that intracellularS100A9 does not play a role in inducing a pro-inflammatory response.Treatment of WT or S100A9 BMDMs with S100A9 protein did not alter IAVreplication status in the corresponding cells (not shown). The inventorsalso observed production of S100A9 following treatment of BMDMs withsynthetic dsRNA (poly-IC). The pro-inflammatory activity of S100A9 wasspecific for IAV and dsRNA (which serves as a replicative intermediateduring IAV infection and induces DDX21/TRIF pathway) since dsRNA(poly-IC) dependent TNF and IL-6 release was significantly diminished inS100A9 KO cells, and treatment of KO cells with purified S100A9 proteinrestored the pro-inflammatory response in poly-IC treated S100A9 KOcells. In contrast, TNF and IL-6 release from S100A9 KO BMDMs was notaffected following imiquimod (which activates TLR7 dependentpro-inflammatory response) treatment. In addition, treatment of WT andS100A9 KO BMDMs with TNF (to induce NF-κB dependent inflammatoryresponse via TNF receptor) revealed similar levels of IL-6 productionfrom both WT and KO cells.

During these studies it was observed that IAV replication (as deducedfrom IAV HA mRNA expression) was significantly reduced in S100A9 KOBMDMs compared to WT cells. This result suggested that althoughextracellular S100A9 plays a critical role in modulatingpro-inflammatory response (FIG. 4D), intracellular S100A9 may beinvolved in negatively regulating antiviral factor expression/productionor it is required for efficient virus infection/replication. This is notsurprising in light of previous reports illustrating differentialfunction of extracellular vs. intracellular S100 proteins.

The inventors observed S100A9 production from IAV-infected BMDMs at 4hours postinfection (FIG. 1C) and that TNF and IL-6 are produced fromIAV-infected BMDMs at 8-12 hours postinfection (not shown); thesecytokines are undetectable at 4 hours postinfection (not shown). Thus,S100A9 secretion and production of early pro-inflammatory mediators(e.g. TNF, IL-6) are temporally regulated during IAV-infection.Therefore, extracellular S100A9 is a key regulator of thepro-inflammatory response during IAV infection.

Extracellular S100A9 promotes apoptosis during IAV infection.Macrophages undergo apoptosis during IAV infection (Hoeve et al. (2012)PLoS One 7: e29443; Huang et al. (2011) Am J Respir Crit Care Med 184:259-268). Several studies have demonstrated that S100A9 has apro-apoptotic function in epithelial cells, muscle cells, andneutrophils (Atallah et al. (2012) PLoS One 7: e29333; Ghavami et al.(2010) Cell Res 20: 314-331; Li et al. (2009) Biochem J 422: 363-372;Viemann et al. (2007) Blood 109: 2453-2460; Seeliger et al. (2003) Am jpathol 163: 947-956), but no apoptosis-inducing activity of S100A9 (orany other S100 proteins) in macrophages has been reported. Since IAVinfection resulted in high levels of S100A9 secretion, the inventorsexamined the ability of extracellular S100A9 to induce apoptosis inmacrophages and the role of secreted S100A9 in apoptotic inductionduring IAV infection.

J774A.1 and MH-S macrophages were treated with purified S100A9 proteinfor 48 and 72 h, then examined the apoptotic status of cells bymonitoring annexin V and PI staining. The apoptosis rate was calculatedbased on the number of annexin V positive/PI negative cells (denotingearly apoptosis)+number of annexin V positive/PI-positive cells(denoting late apoptosis) per total number of cells. It was noted thatapoptosis in S100A9 protein-treated mouse macrophages (FIG. 5A and 5B).The result obtained with annexin V and PI staining was confirmed byperforming TUNEL analysis. Similar results were obtained followingtreatment of human U937 macrophages (not shown).

Since IAV infection triggers S100A9 secretion, the inventors nextexamined whether extracellular S100A9 has a role in apoptosis ofIAV-infected macrophages. J774A.1 macrophages were infected with IAV for48 hours in the presence of either control antibody (control IgG) orS100A9 blocking antibody. Significantly diminished apoptosis (reductionof 27%) occurred in macrophages treated with S100A9 antibody (FIG. 5C).These results were further confirmed by TUNEL analysis. Thus,extracellular S100A9 has a critical function in regulating apoptosis ofIAV-infected macrophages.

The TLR4/MyD88 pathway is required for S100A9-mediated pro-inflammatoryresponse following IAV infection. Previous studies have found thatoptimal TLR4 activation by LPS in bone-marrow cells required theactivity of extracellular S100A9/S100A8 complex (Vogl et al. (2007) NatMed 13: 1042-1049). However, it is not known whether S100A9 aloneactivates TLR4, especially in macrophages. In addition, there has beenno report of DAMP proteins like S100A9 activating PRR signaling duringvirus infection. Therefore, the inventors investigated the role of theTLR4/MyD88 pathway in the macrophage pro-inflammatory response by S100A9alone (in the absence of S100A8), and the function of theS100A9/TLR4/MyD88 pathway in regulating the pro-inflammatory response inIAV-infected macrophages. WT and TLR4 KO BMDMs were incubated withpurified S100A9 protein, and then measured IL-6 (FIG. 6A) and TNF (FIG.6B) levels by ELISA. Drastic loss of IL-6 and TNF production wasdetected in S100A9 protein-treated TLR4 KO BMDMs (FIG. 6A and 6B),indicating that TLR4 is absolutely required for the S100A9-mediatedresponse. Drastic reductions in IL-6 and TNF transcripts occurred inS100A9 protein treated TLR4 KO cells.

Since MyD88 is one of the critical adaptors for activated TLR4, the roleof MyD88 was investigated by using MyD88 KO BMDMs. Incubation of WT andMyD88 KO BMDMs with purified S100A9 protein significantly reducedproduction of IL-6 (FIG. 6C) and TNF (not shown) from MyD88 KO cells.The loss of cytokine protein production was due to reduced TNF and IL-6gene expression in MyD88 KO BMDMs, thus, demonstrating that theTLR4/MyD88 pathway is required for the S100A9-mediated pro-inflammatoryresponse.

The role of TLR4/MyD88 in stimulating the pro-inflammatory responsefollowing IAV infection was studied. After WT, MyD88 KO, and TLR4 KOBMDMs were infected with IAV, IL-6 levels were assessed by ELISA. Thestudy revealed that TLR4/MyD88 is an essential regulator ofpro-inflammatory response during IAV infection, since significantreduction in IL-6 (FIG. 6D) and TNF (FIG. 6E) production was noted inIAV infected TLR4 KO (FIG. 6D and 6E) and MyD88 KO (FIG. 6D) BMDMs.RT-PCR analysis demonstrated diminished expression of IL-6 mRNA in TLR4KO and MyD88 KO BMDMs. Similarly, it was noted that significantreduction in TNF production from IAV-infected TLR4 KO (FIG. 6E) andMyD88 KO (not shown) BMDMs. The observed effect was independent of virusreplication since compared to WT cells, no change in HA mRNA expressionwas noted in TLR4 KO and MyD88 KO cells. Thus, TLR4/MyD88 activation isa key step for inducing the S100A9-mediated pro-inflammatory response.Also, the S100A9/TLR4/MyD88 pathway is a crucial regulator of thepro-inflammatory response during IAV infection.

TLR4/MyD88 pathway is required for apoptosis during IAV infection. Thestudy showed that extracellular S100A9 uses TLR4/MyD88 signaling for thepro-inflammatory response during IAV infection. TLR4 activation has beenassociated with apoptosis induction via various mechanisms, includingactivation of the pro-apoptotic function of NF-κB, modulation oftumor-suppresser expression or function etc. (Yi et al. (2012) PLoS One7: e36560; Equils et al. (2006) J Immunol 177: 1257-1263; De Trez et al.(2005) J Immunol 175: 839-846; Basak et al. (2005) J Immunol 174:5672-5680; Neal et al. (2012) J Biol Chem 287: 37296-37308; Sanchez etal. (2010) Cell Immunol 260: 128-136; Suzuki et al. (2004) Infect Immun72: 1856-1865). To assess the role of TLR4 in S100A9-mediated apoptosis,WT and TLR4 KO BMDMs were treated with purified S100A9 protein for 72hours. Treatment of WT BMDMs with S100A9 protein induced apoptosis (FIG.7A), which was consistent with previous findings. However, significantloss of apoptosis was observed in S100A9 protein-treated TLR4 KO BMDMs(FIG. 7A).

The role of TLR4 and MyD88 was examined in apoptosis induction duringIAV infection. WT and TLR4 KO BMDMs were infected with IAV and evaluatedapoptosis 48 hours later. Apoptosis analysis by annexin V staining (FIG.7B) and TUNEL (FIG. 7C) revealed that while IAV infection resulted inapoptosis of WT macrophages, a significant reduction in apoptosis wasdetected in TLR4 KO cells. Diminished apoptosis was also observed ininfected MyD88 KO BMDMs (FIG. 7D), indicating that MyD88 is alsorequired during this event. Thus, the S100A9/TLR4/MyD88 pathwayconstitutes one of the mechanisms that modulate apoptosis ofIAV-infected cells.

S100A9 expression in IAV-infected mouse respiratory tract. To establishthe in vivo role of S100A9 in regulating innate response during IAVinfection of the airway, S100A9 expression was evaluated and itssecretion in the IAV-infected mouse respiratory tract. Mice wereintratracheally inoculated with IAV and, at 1-6 days postinfection,lungs were harvested. S100A9 mRNA expression in the lungs were analyzedby RT-PCR. S100A9 transcripts were observed in infected lungs but not inlungs from uninfected animals (FIG. 8A), indicating that IAV infectionled to robust induction of S100A9 gene expression. High levels of S100A9protein were detected in the lungs of IAV-infected mice (FIG. 8B).Immunohistochemical analysis of lung sections confirmed the presence ofS100A9 protein in IAV-infected animals (FIG. 8C), while S100A9 wasnearly undetectable in mock-infected lungs. Analysis of bronchoalveolarlavage fluid (BALF) by ELISA confirmed the presence of S100A9 protein inthe airway of IAV-infected animals (FIG. 8D). Thus, IAV infection of therespiratory tract results in induction of S100A9 gene expression andsecretion of S100A9 protein in the airway.

Extracellular S100A9 regulates IAV susceptibility and lung inflammation.Macrophages play a vital role in the innate response to IAV infection byproducing pro-inflammatory mediators that determine the inflammationstatus in the lung (Peschke et al. (1993) Immunobiology 189: 340-355;Herold et al. (2008) J Exp Med 205: 3065-3077; Hoeve et al. (2012) PLoSOne 7: e29443; Huang et al. (2011) Am J Respir Crit Care Med 184:259-268). Moreover, debris from dead cells, originating from apoptosisof immune cells, contributes to airway inflammation (Herold et al.(2008) J Exp Med 205: 3065-3077; Hoeve et al. (2012) PLoS One 7: e29443;Huang et al. (2011) Am J Respir Crit Care Med 184: 259-268; Welliver etal. (2007) J Infect Dis 195: 1126-1136; Hinshaw et al. (1994) J Virol68: 3667-3673; Zhang et al. (2010) Virol J 7: 51; Lu et al. (2010) J GenVirol 91: 1439-1449; Brydon et al. (2005) FEMS Microbiol Rev 29:837-850; Vandivier et al. (2006) CHEST 129: 1673-1682). Sinceextracellular S100A9 acted as a positive regulator of pro-inflammatoryresponse and induced apoptosis, it was hypothesized that extracellularS100A9 exacerbates IAV-associated lung disease. To test this, theinventors used anti-S100A9 blocking antibody, which neutralizesextracellular (secreted) S100A9 protein.

Anti-S100A9 antibody was used instead of doing in vivo studies withS100A9 KO mice because S100 proteins have both intracellular functions(such as cytoskeletal rearrangement, cell metabolism, intracellularcalcium response etc.) and extracellular functions (Hermann et al.(2012) Front Pharmacol 3: 67; Halayko and Ghavami (2009) Can J PhysiolPharmacol 87: 743-755). Since the inventors have elucidated a role ofextracellular (secreted form) S100A9, results from KO mice might notdistinguish whether the observed effects are due to activity ofextracellular S100A9 or intracellular S100A9 function. Most importantlythe studies demonstrated that intracellular S100A9 could be involved innegatively regulating antiviral response or it is required for IAVinfection/replication, since reduced virus replication was noted inS100A9 KO BMDM compared to WT cells. In that scenario, S100A9 KO micemay not serve as an appropriate model to study IAV-inducedpro-inflammatory (and apoptotic) response in vivo, since viral burden inthe lung is directly proportional to the degree of pro-inflammatory (andapoptotic) response (i.e. if there is less viral burden thenconcomitantly reduced pro-inflammatory response and apoptosis willoccur). However, neutralizing the activity of extracellular S100A9 withS100A9 blocking antibody did not alter IAV replication in vitro and invivo. The inventors therefore used anti-S100A9 blocking antibody tospecifically inhibit the activity of extracellular S100A9 in mice. Theinventors have previously shown that anti-S100A9 blocking antibody hasextracellular S100A9 blocking activity (Ryckman et al. (2003) J Immunol170: 3233-3242; Anceriz et al. (2007) Biochem Biophys Res Commun 354:84-89; Cesaro et al. (2012) PLoS One 7: e45478; Simard et al. (2010) JLeukoc Biot 87: 905-914; Simard et al. (2011) J Immunol 186: 3622-3631;Raquil et al. (2008) J Immunol 180: 3366-3374; Vandal et al. (2003) JImmunol 171: 2602-2609. Specifically, intraperitoneal (i.p) injection ofS100A9 blocking antibody inhibited the activity of mouse S100A9 duringS. pneumoniae infection (Raquil et al. (2008) J Immunol 180: 3366-3374).Thus, this antibody is useful to assess the functional role ofextracellular (secreted form) S100A9. Furthermore, similar levels ofS100A9 protein were detected in the BALF of control IgG-treated andS100A9-antibody treated mice. Thus, i.p.-injected anti-S100A9 antibodydid not significantly affect S100A9 protein production in theairway-lumen following IAV infection. As in previous reports, theinventors detected anti-S100A9 antibody (administered i.p.) in lunghomogenate. Thus, anti-S100A9 antibody could effectively blocklung-localized S100A9 during IAV infection (Raquil et al. (2008) JImmunol 180: 3366-3374; Kim et al. (2012) Am J Respir Cell Mol Biol 47:372-378; Toews et al. (1985) Infect Immun 48: 343-349). The clinicalsignificance of utilizing neutralizing antibody is obvious from possiblepassive immunization with S100A9 antibody as a new therapeutic strategyto control lung inflammation and associated lung disease during IAVinfection.

Initially, the inventors investigated the role of secreted S100A9 inregulating IAV susceptibility. For these studies, mice were i.p.injected with either control IgG or anti-S100A9 blocking antibody. Oneday later, mice were infected with IAV via intra-tracheal (I.T)inoculation. Survival of IAV-infected mice was monitored until 8 dayspostinfection. Blocking S100A9 activity significantly reduced themortality of IAV-infected mice (FIG. 9A), demonstrating thatextracellular S100A9 is a key regulator of IAV susceptibility.Extracellular S100A9 also contributes to morbidity since mice treatedwith S100A9 blocking antibody exhibited reduced weight loss upon IAVinfection. Thus, extracellular S100A9 contributes to both IAV-inducedmortality and morbidity.

In addition, inflammation was reduced following inhibition ofextracellular S100A9 activity (FIG. 9B). These results demonstrated thatextracellular S100A9 contributes to the severity of IAV-associated lunginflammation and serves as a critical host factor for heightened IAVsusceptibility and IAV-induced death. The clinical significance of ourresult is borne out by the possibility of passive immunization withanti-S100A9 antibody to reduce the severity of respiratory diseaseassociated with IAV infection.

Extracellular S100A9 controls the pro-inflammatory response inIAV-infected lungs. The inventors have identified extracellular(secreted) S100A9 as a critical regulator of the pro-inflammatoryresponse following IAV infection of macrophages. To examine thephysiological role of secreted S100A9 in lung inflammation, theinventors tested whether intratracheal (I.T.) administration of purifiedS100A9 protein would trigger a pro-inflammatory response in the lungs.Indeed, this led to production of TNF (FIG. 9C) and IL-6 in therespiratory tract due to S100A9-mediated upregulation of TNF and IL-6gene expression in the lung (not shown). The ability of S100A9 proteinto trigger pro-inflammatory mediators in the lung is further reflectedby observing airway inflammation in S100A9 protein administered (viaI.T) mice.

Based on this observation, the role of extracellular S100A9 in airwaypro-inflammatory response following IAV infection was examined. Micewere given i.p. injections of control IgG antibody or anti-S100A9blocking antibody. At ld post-antibody treatment, mice were infectedwith IAV via I.T route. Levels of IL-6 and TNF in the lung were measuredby ELISA. Extracellular S100A9 contributes to production ofpro-inflammatory mediators during infection as evident from reduced TNF(FIG. 9D) and IL-6 levels in the lung of S100A9 antibody treated mice.Reduced pro-inflammatory cytokine production was caused by loss of TNFand IL-6 mRNAs in the lungs of IAV-infected mice treated with S100A9blocking antibody. Diminished pro-inflammatory response is not due toreduced IAV infection, since both control antibody and S100A9 antibodytreated mice exhibited similar IAV infection status (i.e. viral burden).Interestingly, S100A9 antibody could also be utilized as therapeutics tocontrol IAV-associated disease, since administration of S100A9 blockingantibody after IAV infection significantly reduced pro-inflammatoryresponse and lung inflammation.

In order to provide evidence for direct neutralization of S100A9activity in the airway following i.p. administration of S100A9 antibody,S100A9 antibody was administered (via i.p.) to mice and after one day(to exactly mimic IAV infection studies) mice were inoculated withS100A9 protein via I.T route. Significant inhibition in pro-inflammatoryactivity was noted in the presence of S100A9 antibody, which shows thati.p. administered blocking antibody can neutralize S100A9 protein in theairway.

The role of extracellular S100A9 was further validated by conducting exvivo experiment with BALF-associated cells derived from IAV infectedmice administered (via i.p) with either control antibody or S100A9blocking antibody. Significant reduction in IL-6 and TNF production fromBALF cells was observed in S100A9 blocking antibody treated mice (FIG.9E). This result once again validates blocking of S100A9 activity in thealveolar space localized (i.e. present in the BALF) cells. These studiesillustrate the importance of secreted S100A9 in regulatingpro-inflammatory cytokine gene expression and production during IAVinfection of the airway.

Extracellular S100A9 promotes apoptosis in the respiratory tract ofIAV-infected mice. The studies with macrophages have illuminated a vitalrole of extracellular S100A9 in inducing apoptosis of IAV-infectedcells. The inventors have extended those observations in mice toestablish the in vivo physiological relevance of extracellular S100A9 asa regulator of apoptosis. Further, it is known that apoptosissignificantly contributes to IAV infection severity and associated lungdisease. Therefore, reduced apoptosis in IAV-infected S100A9-blockedmice may contribute to reduced susceptibility and diminished airwaydisease (as shown in FIG. 9A and 9B). To examine this possibility, micetreated with control IgG and S100A9 blocking antibody were inoculatedwith IAV via the I.T route. On the third day post-infection, in situTUNEL assay with lung sections were performed to determine the apoptoticstatus of the IAV-infected respiratory tract. The inventors foundsignificantly less apoptosis in the lungs of mice given S100A9 blockingantibody than in the lungs of control mice (FIG. 10A and 10B). Theseresults demonstrated that secreted S100A9 is a pivotal regulator of lungapoptosis following IAV infection.

Methods

Virus, cell culture, mice. Influenza A [A/PR/8/34 (H1N1)] virus wasgrown in the allantoic cavities of 10-day-old embryonated eggs (Sabbahet al. (2009) Nat Immunol 10: 1073-1080; Mgbemena et al. (2012) JImmunol 189: 606-615). Virus was purified by centrifuging two times ondiscontinuous sucrose gradients (Sabbah et al. (2009) Nat Immunol 10:1073-1080; Mgbemena et al. (2012) J Immunol 189: 606-615; Ueba (1978)Acta Med Okayama 32: 265-272). J774A.1 cells were maintained in DMEMsupplemented with 10% fetal bovine serum (FBS), penicillin,streptomycin, and glutamine. U937 cells were maintained in RPMI 1640medium supplemented with 10% FBS, 100 IU/mL penicillin, 100 m/mLstreptomycin, 1 mM sodium pyruvate, and 100 nM HEPES. MH-S cells weremaintained in RPMI 1640 medium supplemented with 10% FBS, 100 IU/mLpenicillin, and 100 m/mL streptomycin. Bone-marrow-derived macrophages(BMDMs) were obtained from femurs and tibias of wild-type (WT) andknock-out mice and were cultured for 6-8 days as described earlier(Sabbah et al. (2009) Nat Immunol 10: 1073-1080; Mgbemena et al. (2012)J Immunol 189: 606-615). Cells were plated on 12-well plates containingRPMI, 10% FBS, 100 IU/mL penicillin, 100 m/mL streptomycin, and 20 ng/mlGM-CSF. Alveolar macrophages were obtained from the broncho-alveolarlavage fluid (BALF) of wild-type C57BL/6 mice. The IAV titer wasmonitored by plaque assay analysis with MDCK cells.

S100A9 KO mice were generated at University of Laval, Quebec, Canada.Other KO mice (TLR4, TLR2, TRAM, TRIF, TIRAP) were originally providedby Dr. Doug Golenbock (University of Massachusetts Medical School,Worcester, Mass.). TLR3 KO, TLR7 KO and MyD88 KO mice were obtained fromJackson Laboratory, Bar Harbor, Me.

Antibodies and proteins. Murine S100A9 neutralizing antibody purifiedIgG from the serum of S100A9 immunized rabbits was generated asdescribed previously (Ryckman et al. (2003) J Immunol 170: 3233-3242;Anceriz et al. (2007) Biochem Biophys Res Commun 354: 84-89). Thisantibody has been successfully used to block the activity ofextracellular mouse S100A9 (Ryckman et al. (2003) J Immunol 170:3233-3242; Anceriz et al. (2007) Biochem Biophys Res Commun 354: 84-89;Cesaro et al. (2012) PLoS One 7: e45478; Simard et al. (2010) J LeukocBiot 87: 905-914; Simard et al. (2011) J Immunol 186: 3622-3631; Raquilet al. (2008) J Immunol 180: 3366-3374; Vandal et al. (2003) J Immunol171: 2602-2609). Human S100A9 antibody was acquired from AbCam,Cambridge, Mass. (goat anti-human S100A9 antibody) and R&D Systems(mouse anti-human antibody). Recombinant human and mouse S100A9 proteinswere generated as previously described (Ryckman et al. (2003) J Immunol170: 3233-3242; Anceriz et al. (2007) Biochem Biophys Res Commun 354:84-89; Cesaro et al. (2012) PLoS One 7: e45478; Simard et al. (2010) JLeukoc Biol 87: 905-914; Simard et al. (2011) J Immunol 186: 3622-3631;Raquil et al. (2008) J Immunol 180: 3366-3374; Vandal et al. (2003) JImmunol 171: 2602-2609). Briefly, full length human S100A9 cDNA wascloned into pET28 expression vector (Novagen, Madison, Wis.). S 100A9protein expression was induced with 1 mM isopropyl β-D-thiogalactoside(IPTG) in E. coli HMS 174 (Boehringer Mannheim, Mannheim, Germany) for16 h at 16° C. After IPTG treatment, the bacteria were centrifuged at5000×g for 10 min and the pellet was re-suspended in PBS [(containingNaCl (0.5 M) and imidazole (1 mM)] and lysed by sonication. Uponcentrifuging the lysate at 55,000×g for 30 min at 4° C., the supernatantwas collected. Recombinant His-Tag S 100A9 was purified by using anickel column. S100A9 bound to the column was incubated with 10 U ofbiotinylated thrombin (Novagen) (for 20 h at room temperature) to freeS100A9 from its His-Tag. Recombinant S100A9 was then eluted with PBS.The digestion and elution processes were repeated one more time tocleave the remaining undigested recombinant proteins, andstreptavidin-agarose (Novagen) was added to remove contaminatingthrombin. Finally, the protein preparation was passed through apolymyxin B-agarose column (Pierce, Rockford, Ill.) to removeendotoxins.

Recombinant proteins were prepared in Hank's buffered salt solution(HBSS) buffer. The absence of endotoxin contamination in antibody andprotein preparations was confirmed using the limulus amebocyte assay(Cambrex).

Reverse transcription-PCR (RT-PCR). Total RNA was extracted using TriReagent (Invitrogen). cDNA was synthesized using a High-Capacity cDNAReverse Transcription Kit (Applied Biosystems). PCR was done using 0.25units of Taq polymerase, 10 pmol of each oligonucleotide primer, 1 mMMgCl₂, and 100 μM deoxynucleotide triphosphates in a final reactionvolume of 25 μl. Following amplification, the PCR products were analyzedon 1.5% agarose gel. Equal loading in each well was confirmed byanalyzing expression of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH). The primers used to detect the indicated genes byRT-PCR were:

GAPDH forward, (SEQ ID NO: 1) 5′-GTCAGTGGTGGACCTGACCT, GAPDH reverse,(SEQ ID NO: 2) 5′-AGGGGTCTACATGGCAACTG, Mouse GAPDH forward,(SEQ ID NO: 3) 5′-GCCAAGGTCATCCATGACAACTTTGG, Mouse GAPDH reverse,(SEQ ID NO: 4) 5′-GCCTGCTTCACCACCTTCTTGATGTC Mouse S100A9 forward,(SEQ ID NO: 5) 5′-GTCCTGGTTTGTGTCCAGGT, Mouse S100A9 reverse,(SEQ ID NO: 6) 5′-TCATCGACACCTTCCATCAA Mouse DDX21 forward,(SEQ ID NO: 7) 5′-GATCCCCCTAAATCCAGGAA, Mouse DDX21 reverse,(SEQ ID NO: 8) 5′-TTCGGAAGGCTCCTCTGTTA Mouse TNF-α forward,(SEQ ID NO: 9) 5′-CCTGTAGCCCACGTCGTAGC, Mouse TNF-α reverse,(SEQ ID NO: 10) 5′-TTGACCTCAGCGCTGAGTTG Mouse IL-6 forward,(SEQ ID NO: 11) 5′-TTGCCTTCTTGGGACTGATGCT, Mouse IL-6 reverse,(SEQ ID NO: 12) 5′-GTATCTCTCTGAAGGACTCTGG IAV HA forward,(SEQ ID NO: 13) 5′-CCCAAGGAAAGTTCATGG, IAV HA reverse, (SEQ ID NO: 14)5′-GAACACCCCATAGTACAAGG

Viral infection of cells. U937 cells, alvelolar macrophages, BMDM, MH-S,and J774A.1 were infected with purified IAV [1 multiplicity of infection(MOI)—2 MOI as indicated] in serum-free, antibiotic-free OPTI-MEM medium(Gibco). Virus adsorption was done for 1.5 h at 37° C., after whichcells were washed twice with PBS. Infection was continued in thepresence of serum containing DMEM or RPMI medium for the specified timepoints.

In some experiments, cells were infected in the presence of 2 ng-10ng/ml control IgG (purified rabbit IgG, Innovative Research, Novi, MI)or 2 ng-10 ng/ml anti-S100A9 blocking antibody. Following virusadsorption, antibodies were added to the cells and the infection wascarried out in the presence of the antibodies. In addition, in someexperiments infection was done in the presence of purified S100A9protein or HBSS buffer (vehicle control). Purified S100A9 protein (5μg/ml) was added to S100A9 KO BMDMs following virus adsorption. Purifiedprotein was present during infection.

siRNA. Control siRNA and mouse DDX21 siRNA were purchased from SantaCruz Biotechnology. MH-S cells were transfected with 40 pmol of siRNAsusing Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, thecells were infected with IAV.

ELISA assay. Medium supernatant and mouse lung homogenate were analyzedfor TNF and IL-6 levels by using a TNF and IL-6 specific ELISA kit(eBioscience, San Diego, Calif.). For S100A9 ELISA, Costar High-Binding96-well plates (Corning, N.Y.) were coated overnight at 4° C. with 800ng/well of purified rabbit IgG against mouse S100A9 or 100 ng/well ofgoat polyclonal human S100A9 antibody (Abcam) diluted in 0.1 M carbonatebuffer, pH 9.6. The wells were blocked with PBST+1% BSA for 1 h at roomtemperature. The samples were added and incubated overnight at 4° C. Theplates were washed three times with PBST and incubated with either goatanti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouseanti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSAfor 2 h at room temperature. The plates were then washed three times inPBST. To detect mouse S100A9, rabbit anti-goat HRP (Bio-Rad) was addedto the plates. To detect human S100A9, goat anti-mouse HRP (Bio-Rad) wasadded. After 1 h incubation at room temperature, the plates were washedthree times with PBST. TMB-S substrate (100 μ/well) (Sigma-Aldrich) wasadded to the plates according to the manufacturer's instructions. TheODs were detected at 450 nm, using a Modulas micro-plate reader.

To detect i.p.-injected S100A9 antibody in the lung homogenate, CostarHigh-Binding 96-well plates were coated overnight at 4° C. with mouseS100A9 protein diluted in 0.1 M carbonate buffer, pH 9.6. The wells wereblocked with PBST+1% BSA for 1 h at room temperature. The lunghomogenate was added and incubated overnight at 4° C. The plates werewashed three times with PBST and goat anti-rabbit HRP (Bio-Rad) wasadded. After 1 h of incubation at room temperature, the plates werewashed three times with PBST. TMB-S substrate (100 μl/well)(Sigma-Aldrich) was added to the plates according to the manufacturer'sinstructions. ODs were detected at 450 nm by using a Modulas micro-platereader.

IAV infection of mice. For survival experiments, 6-8-week oldpathogen-free WT C57BL/6 mice (Jackson Laboratory) were injected i.p.with 2 mg/mouse of either control IgG or anti-S100A9 antibody. One daylater, mice were anesthetized and inoculated via the intratracheal orI.T route with IAV (1×10⁵ pfu/mouse) in 100 μl of PBS (Invitrogen).Control mice were sham-inoculated with 100 μl of PBS. Survival wasmonitored until 8 days postinfection. For pathogenesis assay, mice wereinoculated with IAV (2×10⁴ pfu/mouse via the I.T route) at 1 day afterantibody treatment. At 3 days after infection, lungs and BALF werecollected. Lung tissue sections were used for H&E analysis and in-situTUNEL analysis. Lung homogenate was used for ELISA analysis (for TNF andIL-6). RT-PCR analysis for TNF and IL-6 expression was done with RNAisolated from mouse lungs. BALF was used for Western blotting withS100A9 antibody and S100A9 ELISA analysis.

In some experiments, purified mouse S100A9 protein (15 μg/mouse) dilutedin PBS or HBSS buffer diluted in PBS (vehicle control) was administeredto mice via the I.T route. At 8 h posttreatment, TNF and IL-6 expressionand production in the lung was monitored by RT-PCR and ELISA.

Immunohistochemistry. Lung sections from mock- or IAV-infected mice werestained with goat anti-mouse S100A9 antibody (1:100 dilution) (R&D) for2 h at room temperature. After washing five times with PBS, lungsections were incubated with anti-goat Texas Red (1:50 dilution) (VectorLabs) for 1 h at room temperature. After washing three times with PBS,sections were mounted with DAPI containing mounting solution(Invitrogen). Sections were visualized by fluorescence microscopy.

In-situ TUNEL assay. To study apoptosis in the respiratory tract, TUNELassays were done. Formalin-fixed lungs from IAV-infected mice were used.The TUNEL assay was done using an ApopTag Peroxidase In Situ ApoptosisDetection Kit (Milipore, Mass). Digital images of TUNEL-stained lungsections were examined by light microscopy. Digital images were used tocount the number of TUNEL-positive cells, using Image J software fromNIH) (available via the world wide web at rsbweb.nih.gov/ij/)asdescribed previously (Mgbemena et al. (2012) J Immunol 189: 606-615).For each analysis, an area of 5.39×10² μm×4.09×10² μm of TUNEL-stainedlung section was scanned by Image J software. Gross apoptotic area wasexpressed as pixels per micron. This value was used to calculate thepercentage of the apoptotic area in each analysis. Three IAV- infectedmice treated with control IgG and three IAV-infected mice treated withS100A9 antibody were used. Data were collected from 9 areas per mousefrom each experimental group. The values obtained from the 27 lungsection areas of each experimental group were used for statisticalanalysis.

H&E staining. Hematoxylin and eosin (H&E) staining was performed onparaffin-embedded mouse lung sections. Briefly, slices of lung weresequentially rehydrated in 100% and 95% ethanol followed by xylenedeparaffinization. After rinsing with distilled water, sections werestained with hematoxylin for 8 min and counterstained in eosin for 1 minfollowed by serial dehydration with 95% and 100% ethanol. Sections werethen mounted on coverslips.

Apoptosis assay. Pathogen-associated molecular patterns (PAMPs) triggerhost immune response by activating pattern recognition receptors liketoll-like receptors (TLRs). However, the mechanism whereby severalpathogens, including viruses, activate TLRs via a non-PAMP mechanism isunclear. Endogenous “inflammatory mediators” called damage-associatedmolecular patterns (DAMPs) have been implicated in regulating immuneresponse and inflammation. However, the role of DAMPs ininflammation/immunity IAV-infected and S100A9 protein-treated cells wereexamined for apoptosis by annexin V labeling, using an annexinV/propidium iodide (PI) apoptosis detection kit (BioVision, CA)[75,78,79]. For TUNEL assay cells were grown in cover slips (12 mmdiameter) (Ted-Pella, CA). TUNEL assay with macrophages was performed byusing DeadEnd Colorimetric TUNEL System (Promega, WI). Digital images ofTUNEL-stained macrophages were examined by light microscopy. Digitalimages were used to count the number of TUNEL-positive cells using ImageJ software (please see above). At least eight different fields werecounted for each cover slip and two cover slips (duplicate) wereexamined for each experiment. Furthermore, each experiment was repeatedindependently three times.

Example 2

S100A9 blocking antibody reduces RSV viral titer following RSVinfection. Mice were infected with respiratory syncytial virus (RSV) inthe presence of either control antibody (IgG) or S100A9 blockingantibody (S100A9 Ab). At 3 days post-infection, the lungs of the micewere isolated and the RSV infectious virus titer was evaluated in theairways by performing plaque assay analysis with lung homogenate.

As shown in FIG. 11, a reduced RSV viral titer was observed in S100A9 Abtreated mice. This result surprisingly indicated that S100A9 Ab maypossess anti-viral properties that can reduce RSV infection in therespiratory tract of subjects. Accordingly, S100A9 Ab can be utilized asa therapeutic agent to combat RSV infection, since it may possessanti-viral activity and can reduce RSV burden in the airway of aninfected subject.

Further experiments with macrophages will be conducted to elucidate themechanism that may contribute to reduced RSV infection followingblocking of extracellular S100A9 with S100A9 Ab, for example theexamination of type I interferon (interferon-beta) response inmacrophages and infected mice following S100A9 Ab administration giventhat interferon-beta is an essential anti-viral cytokine that restrictsRSV infection.

Example 3

S100A9 blocking antibody reduces IL-6 production following RSVinfection. A mouse alveolar macrophage cell line (MH-S) was infectedwith RSV in the presence of control IgG or S100A9 blocking antibody(S100A9 Ab). Thereafter, IL-6 production from the cells was assessed viaELISA.

As shown in FIG. 12, treatment of MH-S cells with S100A9 Ab resulted ina reduction in IL-6 production following RSV infection. Pro-inflammatorycytokines such as IL-6 play a major role in exaggerating inflammationand pneumonia manifestation during RSV infection. This exampledemonstrates that blocking extracellular S100A9 protein with S100A9 Abcan diminish inflammation during RSV infection. Further studies with RSVinfected mice will determine if treatment with S100A9 Ab can reduce RSVassociated airway disease pathogenesis.

1. A method of treating a pathogen-induced lung inflammation in asubject, said method comprising the step of administering an anti-S100A9antibody to the subject in need of thereof.
 2. The method of claim 1,wherein the pathogen is a virus.
 3. The method of claim 2, wherein thevirus is a respiratory virus.
 4. The method of claim 3, wherein therespiratory virus is an influenza virus.
 5. The method of claim 4,wherein the influenza virus is influenza A.
 6. The method of claim 5,wherein influenza A is an influenza A serotype selected from the groupconsisting of H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3,H10N7, and H7N9.
 7. The method of claim 3, wherein the respiratory virusis respiratory syncytial virus (RSV).
 8. The method of claim 1, whereinthe method further comprises administering a second therapeutic agent inconjunction with the anti-S100A9 antibody, wherein the secondtherapeutic agent is an antiviral drug.
 9. The method of claim 8,wherein the second therapeutic agent is selected from the groupconsisting of amantadine, oseltamivir, zanamivir, ribavirin, andpalivizumab.
 10. The method of claim 1, wherein the anti-S100A9 antibodyis administered to the subject via a route selected the group consistingof inhalation to the respiratory system, instillation in the respiratorysystem, intraperitoneal injection, and intravenous injection.
 11. Themethod of claim 1, wherein the anti-S100A9 antibody is a humanizedanti-S100A9 antibody.
 12. The method of claim 1, wherein the anti-S100A9antibody is an antibody fragment or an antibody conjugate.
 13. A methodof treating a respiratory virus infection in a subject, said methodcomprising the step of administering an anti-S100A9 antibody to thesubject in need of thereof.
 14. The method of claim 13, wherein therespiratory virus is RSV.
 15. The method of claim 14, wherein theadministration of the anti-S100A9 antibody reduces RSV infection in therespiratory tract of the subject, and wherein the RSV infection isreduced via a decrease in viral titer.
 16. The method of claim 13,wherein the method further comprises administering a second therapeuticagent in conjunction with the anti-S100A9 antibody, wherein the secondtherapeutic agent is a second antiviral drug.
 17. The method of claim16, wherein the second therapeutic agent is selected from the groupconsisting of amantadine, oseltamivir, zanamivir, ribavirin, andpalivizumab.
 18. The method of claim 13, wherein the anti-S100A9antibody is administered to the subject via a route selected the groupconsisting of inhalation to the respiratory system, instillation in therespiratory system, intraperitoneal injection, and intravenousinjection.
 19. The method of claim 13, wherein the anti-S100A9 antibodyis a humanized anti-S100A9 antibody.
 20. The method of claim 13, whereinthe anti-S100A9 antibody is an antibody fragment or an antibodyconjugate.